Diana Bellavia

Constitutive activation of NF‐κB and T‐cell leukemia/lymphoma in Notch3 transgenic mice

The multiplicity of Notch receptors raises the question of the contribution of specific isoforms to T‐cell development. Notch3 is expressed in CD4−8− thymocytes and is down‐regulated across the CD4−8− to CD4+8+ transition, controlled by pre‐T‐cell receptor signaling. To determine the effects of Notch3 on thymocyte development, transgenic mice were generated, expressing lck promoter‐driven intracellular Notch3. Thymuses of young transgenics showed an increased number of thymocytes, particularly late CD4−8− cells, a failure to down‐regulate CD25 in post‐CD4−8− subsets and sustained activity of NF‐κB. Subsequently, aggressive multicentric T‐cell lymphomas developed with high penetrance. Tumors sustained characteristics of immature thymocytes, including expression of CD25, pTα and activated NF‐κB via IKKα‐dependent degradation of IκBα and enhancement of NF‐κB‐dependent anti‐apoptotic and proliferative pathways. Together, these data identify activated Notch3 as a link between signals leading to NF‐κB activation and T‐cell tumorigenesis. The phenotypes of pre‐malignant thymocytes and of lymphomas indicate a novel and particular role for Notch3 in co‐ordinating growth and differentiation of thymocytes, across the pre‐T/T cell transition, consistent with the normal expression pattern of Notch3.

Combined expression of pTα and Notch3 in T cell leukemia identifies the requirement of preTCR for leukemogenesis

Notch receptors are conserved regulators of cell fate and have been implicated in the regulation of T cell differentiation and lymphomagenesis. However, neither the generality of Notch involvement in leukemia, nor the molecules with which Notch may interact have been clarified. Recently, we showed that transgenic mice expressing the constitutively active intracellular domain of Notch3 in thymocytes and T cells developed early and aggressive T cell neoplasias. Although primarily splenic, the tumors sustained features of immature thymocytes, including expression of pTα, a defining component of the pre T cell receptor, known to be a potent signaling complex provoking thymocyte survival, proliferation, and activation. Thus, enforced expression of Notch3, which is ordinarily down-regulated as thymocytes mature, may sustain pre T cell receptor expression, causing dysregulated hyperplasia. This hypothesis has been successfully tested in this article by the observation that deletion of pTα in Notch3 transgenic mice abrogates tumor development, indicating a crucial role for pTα in T cell leukemogenesis. Parallel observations were made in humans, in that all T cell acute lymphoblastic leukemias examined showed expression of Notch3 and of the Notch target gene HES-1, as well as of pTα a and b transcripts, whereas the expression of all these genes was dramatically reduced or absent in remission. Together, these results suggest that the combined expression of Notch3 and pTα sustains T cell leukemogenesis and may represent pathognomonic molecular features of human T-ALL.

Expression of Activated Notch3 in Transgenic Mice Enhances Generation of T Regulatory Cells and Protects against Experimental Autoimmune Diabetes

Thymic-derived dysregulated tolerance has been suggested to occur in type 1 diabetes via impaired generation of CD4+CD25+ T regulatory cells, leading to autoimmune β cell destruction. In this study, we demonstrate that Notch3 expression is a characteristic feature of CD4+CD25+ cells. Furthermore, streptozotocin-induced autoimmune diabetes fails to develop in transgenic mice carrying the constitutively active intracellular domain of Notch3 in thymocytes and T cells. The failure to develop the disease is associated with an increase of CD4+CD25+ T regulatory cells, accumulating in lymphoid organs, in pancreas infiltrates and paralleled by increased expression of IL-4 and IL-10. Accordingly, CD4+ T cells from Notch3-transgenic mice inhibit the development of hyperglycemia and insulitis when injected into streptozotocin-treated wild-type mice and display in vitro suppressive activity. These observations, therefore, suggest that Notch3-mediated events regulate the expansion and function of T regulatory cells, leading to protection from experimental autoimmune diabetes and identify the Notch pathway as a potential target for therapeutic intervention in type 1 diabetes.

Notch and NF-kB signaling pathways regulate miR-223/FBXW7 axis in T-cell acute lymphoblastic leukemia.

Notch signaling deregulation is linked to the onset of several tumors including T-cell acute lymphoblastic leukemia (T-ALL). Deregulated microRNA (miRNA) expression is also associated with several cancers, including leukemias. However, the transcriptional regulators of miRNAs, as well as the relationships between Notch signaling and miRNA deregulation, are poorly understood. To identify miRNAs regulated by Notch pathway, we performed microarray-based miRNA profiling of several Notch-expressing T-ALL models. Among seven miRNAs, consistently regulated by overexpressing or silencing Notch3, we focused our attention on miR-223, whose putative promoter analysis revealed a conserved RBPjk binding site, which was nested to an NF-kB consensus. Luciferase and chromatin immunoprecipitation assays on the promoter region of miR-223 show that both Notch and NF-kB are novel coregulatory signals of miR-223 expression, being able to activate cooperatively the transcriptional activity of miR-223 promoter. Notably, the Notch-mediated activation of miR-223 represses the tumor suppressor FBXW7 in T-ALL cell lines. Moreover, we observed the inverse correlation of miR-223 and FBXW7 expression in a panel of T-ALL patient-derived xenografts. Finally, we show that miR-223 inhibition prevents T-ALL resistance to γ-secretase inhibitor (GSI) treatment, suggesting that miR-223 could be involved in GSI sensitivity and its inhibition may be exploited in target therapy protocols.

Notch signaling and diseases: An evolutionary journey from a simple beginning to complex outcomes

Notch signaling pathway regulates a wide variety of cellular processes during development and it also plays a crucial role in human diseases. This important link is firmly established in cancer, since a rare T-ALL-associated genetic lesion has been initially reported to result in deletion of Notch1 ectodomain and constitutive activation of its intracellular region. Interestingly, the cellular response to Notch signaling can be extremely variable depending on the cell type and activation context. Notch signaling triggers signals implicated in promoting carcinogenesis and autoimmune diseases, whereas it can also sustain responses that are critical to suppress carcinogenesis and to negatively regulate immune response. However, Notch signaling induces all these effects via an apparently simple signal transduction pathway, diversified into a complex network along evolution from Drosophila to mammals. Indeed, an explanation of this paradox comes from a number of evidences accumulated during the last few years, which dissected the intrinsic canonical and non-canonical components of the Notch pathway as well as several modulatory extrinsic signaling events. The identification of these signals has shed light onto the mechanisms whereby Notch and other pathways collaborate to induce a particular cellular phenotype. In this article, we review the role of Notch signaling in cells as diverse as T lymphocytes and epithelial cells of the epidermis, with the main focus on understanding the mechanisms of Notch versatility.

Pre‐TCR‐triggered ERK signalling‐dependent downregulation of E2A activity in Notch3‐induced T‐cell lymphoma

Notch and basic helix–loop–helix E2A pathways specify cell fate and regulate neoplastic transformation in a variety of cell types. Whereas Notch enhances tumorigenesis, E2A suppresses it. However, whether and how Notch and E2A interact functionally in an integrative mechanism for regulating neoplastic transformation remains to be understood. It has been shown that Notch3‐induced T‐cell leukaemia is abrogated by the inactivation of pTα/pre‐T‐cell antigen receptor (pre‐TCR). We report here that Notch3‐induced transcriptional activation of pTα/pre‐TCR is responsible for the downregulation of E2A DNA binding and transcriptional activity. Further, the E2A messenger RNA and protein levels remain unaltered but the E2A inhibitor Id1 expression is augmented in thymocytes and T lymphoma cells derived from Notch3 transgenic mice. The increase in Id1 expression is achieved by pre‐TCR‐induced extracellular‐signalling‐regulated kinase 1/2. These observations support a model in which the upregulation of pre‐TCR signalling seems to be the prerequi‐site for Notch3‐induced inhibition of E2A, thus leading to the development of lymphoma in Notch3 transgenic mice.

Notch3 and the Notch3-upregulated RNA-binding protein HuD regulate Ikaros alternative splicing

Constitutive activation of the transmembrane receptor, Notch3, and loss of function of the hematopoietic transcription repressor, Ikaros (IK), play direct roles in T‐cell differentiation and leukemogenesis that are dependent on pre‐T‐cell receptor (pre‐TCR) signaling. We demonstrate the occurrence of crosstalk between Notch3 and IK that results in transcriptional regulation of the gene encoding the pTα chain of the pre‐TCR. We also show that, in the presence of the pre‐TCR, constitutive activation of Notch3 in thymocytes causes increased expression of dominantnegative non‐DNA‐binding IK isoforms, which are able to restrain the IK inhibition of Notch3s transcriptional activation of pTα. This effect appears to be mediated by Notch3s pre‐TCR‐dependent upregulation of the RNA‐binding protein, HuD. Notch3 signaling thus appears to play a critical role in the diminished IK activity described in several lymphoid leukemias. By exerting transcription‐activating and transcription‐repressing effects on the pTα promoter, Notch3 and IK cooperate in the fine‐tuning of pre‐TCR expression and function, which has important implications for the regulation of thymocyte differentiation and proliferation.

Notch3: from subtle structural differences to functional diversity.

The Notch3 gene was identified, at the beginning of 90s, as the third mammalian Notch and was initially reported as being expressed in proliferating neuroepithelium. Since then, increasing evidence has demonstrated a number of structural and functional differences between Notch3 and both Notch1 and Notch2, which exhibit the highest structural similarity among the four mammalian Notch receptors. Possibly due to its more restricted tissue distribution, targeted deletion of murine Notch3 does not lead to embryonic lethality as is observed with targeted deletion of Notch1 and Notch2. However, genetic mutation, amplification and deregulated expression of Notch3 have been correlated with the disruption of cell differentiation in transgenic mice and to development of diseases in mice and humans. This review discusses the possible relationships between the structural differences and the nonredundant roles that Notch3 plays in the pathogenesis of the human disease cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy and in the regulation of murine T-cell differentiation and leukemogenesis.

PKC theta mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia.

Protein kinase (PK)Cθ is a critical regulator of mature T-cell activation and proliferation, being implicated in TCR-triggered nuclear factor (NF)-κB activation and providing important survival signals to leukemic T cells. We previously showed that overexpression of pTα/pre-TCR and constitutive activation of NF-κB characterize the T-cell leukemia/lymphoma developing in Notch3-IC transgenic mice. We report here that PKCθ is a downstream target of Notch3 signaling and that its activation and membrane translocation require a functional pre-TCR in order to trigger NF-κB activation in thymocytes and lymphoma cells of transgenic mice. Furthermore, deletion of PKCθ in Notch3-IC transgenic mice reduces the incidence of leukemia, correlating with decreased NF-κB activation. This paper therefore suggests that PKCθ mediates the activation of NF-κB by pre-TCR in immature thymocytes and contributes to the development of Notch3-dependent T-cell lymphoma.

Notch3 and canonical NF-κB signaling pathways cooperatively regulate Foxp3 transcription

Notch3 overexpression has been previously shown to positively regulate the generation and function of naturally occurring regulatory T cells and the expression of Foxp3, in cooperation with the pTα/pre-TCR pathway. In this study, we show that Notch3 triggers the trans activation of Foxp3 promoter depending on the T cell developmental stage. Moreover, we discovered a novel CSL/NF-κB overlapping binding site within the Foxp3 promoter, and we demonstrate that the activation of NF-κB, mainly represented by p65-dependent canonical pathway, plays a positive role in Notch3-dependent regulation of Foxp3 transcription. Accordingly, the deletion of protein kinase Cθ, which mediates canonical NF-κB activation, markedly reduces regulatory T cell number and per cell Foxp3 expression in transgenic mice with a constitutive activation of Notch3 signaling. Collectively, our data indicate that the cooperation among Notch3, protein kinase Cθ, and p65/NF-κB subunit modulates Foxp3 expression, adding new insights in the understanding of the molecular mechanisms involved in regulatory T cell homeostasis and function.